Everninomicin antibiotics and method for the manufacture thereof



March 3, 1970 LUEDEMANN ETAL 3,499,078

EVERNINOMICIN ANTIBIOTICS AND METHOD FOR THE MANUFACTURE THEREOF FiledSept. 25, 1968 2 Sheets-Sheet 2 FIG.3

FIG.4

GEORGE M. LUEDEMANN MARVIN J. WEINSTEIN -5' I m A,

AGEN

United States Patent "cc 3,499,078

Patented Mar. 3, 1970 3 499 078 sion, Peoria, Ill. where it was assignedthe number NRRL EVERNINOMICIN ANTIBIOTICS AND METHOD 2972- FOR THEMANUFACTURE THERE F The antibiotics of th1s 1nvent1on are also producedby George M. Luedemann, Glen Ridge, and Marvin J. Weina color variant ofMicromono-spora carbonacea var. carstein, East Brunswick, N.J.,assignors to Schering Cor- 5 bonacea which was obtained from the samegeneral locale p f' b Bloomfield, corporation Of New Jersey (Clean,N.Y.). The variant is herein designated Micro- X S i EgZ -g 13 glill l si i l l g- 1 :2 3 monospora carbonacea var. aurantiaca thus denoting itspr. er. 0. uy an e. h

No. June 1966. This applicatlonasept. 25, orange color A visible cultureof th1s varlant as been deposited and made part of the stock culturecollec- 55 6 (307g 11/00. (321d 9/00 10 tion of the United StatesDepartment of Agriculture, s CL 9 Claims Northern Utilization Researchand Development Division,

Peoria, 111., where it was assigned the number NRRL 2997. In general,the properties of the two variants are ABSTRACT OF THE DISCLOSUREvirtually identical except for their difierence in color b b d a andother minor characterlstics described herein. Con- Mlcmmonospom. W caronacea an sequently, where no specific variant is being discussedV.anant unmet-2M1 omonmp 0m carbonaiiea f the variants will be referredto generically as Micromonotzaca, when Sub ected to submerged aerobicfermentation spam carbonacea or M. carbonacem linder l i i p q t Producea mlxfure of at leait M. carbonacea var. carbonacea is characterized bythe SIX sa.ld l i m belig de.s1.gnated i i' development of coal-blackareas of spore accumulation i i l i q evemmqmlclg evermm' on anotherwise orange vegetative mycelial colony when nomicin D, evermnomicinE and evernmomlcm F, respecgrown on 05% yeast extract, 1% dextrose, 0.1%CaCOB tlvely- 1.5% agar and incubated for 20 days at 28 C. The spores ofM. carbonacea var. carbonacea tend to be This application is acontinuation-in-part f pr r oblong ellipsoidal and the average dimensionof the Pending applications 5611 567,811, filed 111116 13, 1966 maturespore is 1.0 by 1.5a. The fully mature spores abandoned); 5 filed y 24,1963 when microscopically viewed by transmitted light are abandoned); 2fi y 16, 1962 dark in color. Sporophores appear in large concentrationsabandoned); and 5611 184,484, filed P 2, in particular areas of thevegetative mycelium rather 1962 (now abandoned). 30 than randomlythroughout the entire mycelial length. The This invention relates tocompositions of matter which vegetative mycelium does not normallyappear to break have antibiotic properties and to methods for the marmupinto polymorphic elements (fragments) but retains facture and usethereof. its integrity until eventual autolysis. The vegetative my- Thenovel antibiotics f his i n n a fofmtad y celium averages 0.6 1. indiameter, is non-acid fast, and cultivation under controlled COHditiOHSOf hitherto 1111- gra n-positive, The Organism digests certain types ofdescribed Species of the genus MiCYOmO'IZ-YPOM 0f the protein andstarch, is aerobic, and grows well between der Actinomycetales. 16-37 C.but not at 50 C. or above. While the colony The antibiotlcs of theinstant invention adversely affect for d orange color ith b i hbh k withe growth of a Wide Variety of microorganisms Such as are typical ofthe fresh soil isolate, the characteristics species Of Staphylococcus,Escherichia, Klebsiella, Sal- 40 may be temporarily or permanently lostin strains bmonella and the like. They are useful, therefore, as a t i dby repeated i ol tio a d tr f bacteriostatic component iS wash solutionssuch 38 are Coloney bservations were made 01'1M carbanacga var, used toClean Surgical q p laboratory glassware, carbonacea after 14 daysincubation at 242.6 C. in the military mess halls and the like. Further,the instant antid i d di I d ibi th 1 f r ti blOtiCS manifest a broadrange of antibacterial properties (Table the following ystem andreferences are emp in ViVO utilization, as is describedhereinbelowployed: The color designation consists of two designates. THEMICROORGANISM The first is a color name taken from the Descriptive Th f1 f th f th Color Name Dictionary, by Taylor, Knoche andGranimcroorggmims i H or e.prepara 0 e ville, published by the ContainerCorporation of America,

antibiotics of this lnvention are species of M1crom0n0- 1950 (Us A) witha color chi number Cortes ondi spora, isolated from a soil sampleobtained in Clean, New p p Hg York, USA. Applicants designate one suchorganism to the color name; said chip nurflijer taken from, theMzlcromonospora carbonacea nov. sp. or more accurately Color Manualedmon Pubhshed Micromonospora carbonacea var. carbonacea to denote bythe columnar corPoranon of Amenca The its black color. A variableculture of the living organism secfmd deslgnate, Conslsts of acolor'nalne and has been deposited and made part of the stock cultureWhlch refers to the synonym Of Real" y y found In collection of theUnited States Department of Agriculture, the National Bureau ofStandards, Circular v m- Northern Utilization Research and DevelopmentDiviher 1, 1955 (U.S.A.).

TABLE I.COLONY COLOR CHARACTERISTICS OF M. Carbonacea var. CarbonuceaColony Observations Medium Macroscopic Microscopic 0.1% NZ amine Type A,1 1% dextrose, 1.5% agar- N o aerial mycelium; colony flat, moist;growth Myceliurn long, branched, regular, nonseptatei fair; nodifiusible pigment produced; surface: 0.6;. in diameter. Sporesabundant, oblong (periphery) brite orange-g4NA; vivid orangotoellipsoidal, 1.0 x 1.5 dark in color, borne 48 (center) brownish-black;reverse: id. 011 clusters of sporophores.

3% NZ amine TypeA, 1% dextrose, 1.5% agar No aerial mycelium; colonyraised, ridged; Mycelium long, branched, regular, nonscptate, growthgood; no diflusible pigment produced; 0.6 in diameter. Spores oblong toellipsoidal surface: term cotta-gSPE strong brown-55; 1.0 x 1.5;, darkin color, borne singly on single reverse: id. Sporophores.

Shetfield Chemical Company, Norwich, New York.

Although the invention as set forth herein describes the use of the M.ca'rbonaceai var. carbonacea to produce certain antibiotics describedherein, it is to be understood that the process aspect of the inventionis not so limited but includes the hereinabove mentioned color variant.It also includes mutants and variants produced from the describedorganisms by mutating agents such as high frequency radiation (X-ray,ultraviolet), actinophages and nitrogen mustards.

To isolate the microorganism, a portion of the soil sample is shaken insterile distilled water and after making suitable dilutions, thesuspension is plated on screen agar medium comprising 0.5% tryptose,2.0% soluble starch, 0.3% calcium propionate, 2.0% agar all in distilledwater.

The cultures are tested for antibiotic activity by first growing for upto 60 days at 26 C. in a medium comprising 0.3% beef extract, 0.5%tryptose, 0.1% dextrose, 2.4% soluble starch, 0.5% yeast extract, 1.5%agar, all in tap water. The whole aqueous agar is then extracted withbutanol and the butanol-water extract is concentrated. S'uificientantibiotic is extracted by the butanol-water mixture to provide aconcentrate which, by standard disc test inhibits the growth ofStaphylococcus aureus and Bacillus subtilis. Antibiotic activity againstthe same organisms is observed after growth in submerged fermentationfor 96 hours in aqueous medium such as one containing 0.5 yeast extract,0.1% fish solubles, 0.1% calcium carbonate, 0.1% spray dried corn steepliquor, 3.0% lactose.

Colonies incubated for 14 days at 26 C. on various media displayed thefollowing growth and colony characteristics: it grows poorly onglucose-asparagine agar, liquifies gelatin, slowly digests milk,utilizes sucrose, hydrolyzes starch, reduces nitrates to nitriates,exhibits good growth at 16-37 C. but no growth at 50 C. and is aerobicin its requirements.

Growth and colony characteristics have been observed in media commonlyemployed for Streptomyces determinations. The growth characteristics onsuch media are as follows: Potato slicefair, carrot slice-poor, Czapeksagarnone, tyrosine agar (observations made at 2, 7 and 14 days)-fairwith no ditfusible pigments, peptone-iron agar (observations at 2, 7 and14 days)-fair with no color reaction, bromocresol purple milkfair withpartial digestion. In Table II there is presented the growth and colonycolor characteristics observed on various media. The color systememployed is the same as that in Table I.

TABLE l'L-GROWTH AND COLOR CHARACTERISTICS OF M.

C'EA var. CARBONACEA gen source, all in distilled water. With asparagineor glutamic acid, growth is poor while no growth is observed in mediacontaining ammonium or sodium nitrate. With 1% NZ Amine Type A growth isfair with the colony raised and ridged. The colony color is russetorange-g4PC; deep orange-51 (with slight brownish-black mottling). Whenthe nitrogen source is 0.5% Difco yeast extract, growth is fair, thecolony is raised and ridged with peripheral color being orangeg5LA;strong orangeand center color being mottled brownish-black.

M. carbonacea var. aurantiaca is taxonomically similar to M. carbonacca'var. carbonacea with the following exceptions: This variant fails toreduce nitrate, occasionally produces a yellow diifusible pigment whengrown in a carbohydrate medium of mannose or xylose, and exhibits alimited product of spores. The colony color on most agar media is orangeturning orange-brown on aging. Occasionally, small black areas,consisting almost entirely of dark colored spores appear on the orangesurface. The orange areas are composed mainly of vegetative mycelium. OnBennetts agar, growth of the variant is good, raised, with colony colorbeing burnt-orange-g5NC; strong reddish orange35. On tomato paste,oatmeal agar (containing 0.1% sodium carbonate) growth is fair andraised with colony color being bittersweetg5PC; deep orange-51.

THE ANTIBIOTIC M. carbonacea by the fermentation methods describedherein, produces antibiotic compositions of matter. After thefermentation, when the mycelium is separated from the broth, theantibiotic substances are found primarily in the broth and are separatedtherefrom by the methods described herein. By paper chromatographicstudies, it appears that there are a number of diiferent antibioticsubstances produced, six of which are hereinafter identified aseverninomicin A, B, C, D, E and F respectively (formerly R-451A, B, C,D, E and F respectively), the resolution of which is dependent upon themethod of partition chromatography employed.

Degradation studies on the antibiotics of this invention show that atleast three members of the group (everninomicin B, D and F) containdichloroisoeverninic acid as an integral part of its molecularstructure. Consequently, the group was collectively named everninomicin.The three above-named members of the group appear to contain theprincipal antibiotic properties, therefore, it is within them CARBON!!-Medium plus 0.1% CaGO Observed growth and colony color.

black) Emersons Agar. Growth fair, raised. Colony color: ten-a.cotta-g5PE;

strong brown-55.

Tomato Paste-Oatmeal Agar. Growth good, ridged. Colony color: periphery:0range gfiLA; strong orange-50 center: brownish-black.

Glucose Yeast Extract Again-.- Growth fair, ridged. Colony color: russetorange-g4NG;

deep orange-51.

M. carbonacew is capable of utilizing various carbon and nitrogensources. For determination of carbon utilization a visual estimate ismade of its growth on agar plates in media consisting of 0.5 yeastextract, Difco (Difco Laboratories, Inc., Detroit, Mich), 1.5% agar and1% of the test carbohydrate all in distilled Water. Observable growth isgood in media containing the following carbohydrate component:arabinose, glucose, galactose, lactose, levulose, mannose, starch,sucrose, or xylose but poor in media containing one of the following:raflinose, rhamnose, inositol, mannitol or sorbitol. (Poor growth isobserved in a control medium containing only 0.5 yeast extract.)

Nitrogen utilization is similarly determined by visual estimate ofgrowth on agar plates in a medium consisting of gl co 1. agar and 1% ofthe particular nitrothat this invention is primarily but not exclusivelyconcerned.

In order to form the antibiotic substances, M. carbonacea is grown at asuitable temperature of 25 C. to 40 C., under submerged aerobicconditions in an aqueous nutrient medium containing an assimilablecarbon and nitrogen source. Suitable nitrogen sources include bothorganic and inorganic nitrogen, preferably the former, such as soybeanmeal, peptones and the like. Suitable carbon sources includecarbohydrates such as starch, dextrin, sugars and the like.

The fermentation is carried out for about 60 to 72 hours at a pH ofabout 6 to 8. At about the end of this period, peak antibioticproduction has been attained. Since most of the activity resides in thebroth, the my celium is removed by filtration and discarded. Theantibiotic substances are separated from the broth by solvent extractionwith an immiscible organic solvent. In general, such solvents as thechlorinated alkanes, ethers, esters, aromatic hydrocarbons and the likemay be used. Exemplary of the foregoing are such solvents as methylenechloride, chloroform, ethyl ether, dibutyl ether, ethyl acetate, butylacetate, benzene, toluene and the like.

Resolution into components is effected by partition chromatography,essentially as described herein. The antibiotic substances are firstisolated from the broth-extraction solvent by evaporation of the solventto a residue. The residue is examined paper chromatographically whichserves as a guide for partition chromatography (Table III). The paperchromatograms are run in different solvent system and R values for thecomponents are determined bioautographically. Bioautography utilizes themethod of developing and drying a paper chromatogram which then isoverlaid on an agar plate seeded with S. aureus. After a contact time of15 minutes, the paper is removed from the plate which is then incubatedat 37 C. for 16-20 hours. Observations of the location of inhibitionzones permits determination of R; values of the antibiotically activecomponents.

In the following Table III are shown the various solvent systemsemployed and the R values or R, values determined for the components. Ineach system, descending solvent was employed:

TABLE III.CHROMATOGRAPHIC STUDIES ON ANT I BIOTIC SUBSTANCES PRODUCED BYFERMENTATION Solvent System Everninomicin R; Value Benzene, 10 ptsPetroleum ether (30-60 0.), 2.5 pts Acetone, pts Developing time, 1% hrsToluene, 20 pts n-Butanol, 1.5 pts Distilled water, 7.0 pts- Petroleumether (3060 0.), 1.5 pts Developing time, 4% hrs III.

Ligroin (90120 (3.), 3 pts Butyl acetate, 7 pts. J

B E F Rt Value The above solvent mixture is saturated with a 15% aqueoussolution of 4-chloro-2-methylphenoxyacetic acid sodium salt.

In the examples which follow are illustrated suitable methods forfermenting M. carbonacea, extracting the antibiotic substances andresolving the antibiotic substances into components. Theproceduresassociated with the tank fermentation and work-up utilize a diatomaceousearth column chromatography which effectively resolves the antibioticsubstances and allows for the separation and isolation of evernionomicincomponents B, D and E.

In some of the examples which follow, an assay value of the producedantibiotic is expressed in terms of units per milligram as an indicationof activity. The assay is effected microbiologically by a standardcup-type agar diffusion assay technique using S. aureus (A.T.C.C. 6538P)as tests organism. A reference curve is prepared by plotting thedosage-response of the antibiotic diluted in phosphate buffer at pH 8 ina medium consisting of:

Percent Peptone 0.6 Pancreatic digest of casein 0.4 Yeast extract 0.3Beef extract 0.15 Dextrose 0.15 Agar 1.5 pH, 6.6

A suspension of the assay organism (S. aureus A.T.C.C. 65381 isstandardized to provide 20% transmission at 660 my in a colorimeter. Thepotency of the sample is determined from the reference curve andexpressed in terms of units per milligram (a unit being that amount oftest substance required to produce a 15 mm. zone of inhibition with asteel cylinder of 6.5 mm. outside diameter.).

The organism M. carbonacea is mentioned herein as the producer of theherein described antibiotics. It is to be understood, however, that thisis done merely for clarity in reading and understanding thespecification. It is thus to be understood that in the foregoingdiscussion and in the examples which follow, it is intended to indicatethat where M. carbonacea is mentioned as the anti biotic producingmicroorganism that its variant, M. carbonacea var. aurantz'aca and otherequivalent organisms, may be equally utilized to yield essentially thesame result.

EAMPL-E 1 Tank fermentation of M. carbonacea Germinati nstage.-Aseptically add a lyophilized culture of M. carbonacea to a 300ml. shake flask containing 100 ml. of the following sterile growthmedium:

Bacto-beef extract gm 3 Tryptose gm 5 Dextrose gm 1 Starch (soluble) gm24 Yeast extract gm 5 Calcium carbonate gm 1 Tap water ml 1000 Incubatethe flask and its contents for 4 days at 35 C. (or until goodgermination is obtained) on a rotary shaker (2 r.p.m., 2 inch stroke).

lPre-seeding stage.To each of three 300 ml. shake flasks containing ml.of the aforementioned sterile growth medium, add 5 ml. of the inoculumfrom the germination stage. Incubate the flasks and their contents for72 hours at 30 C. on the rotary shaker.

Inoculum preparati n stage.-Transfer 25 ml. inoculums (from thepre-seeding stage flasks) to each of ten 2-liter flasks, each containing500 ml. of the sterile growth medium utilized for germination. Incubatethe flasks and contents for 72 hours at 30 C. on a rotary shaker (280r.p.m., 2" stroke). Pool the contents of the flasks and asepticallytransfer the broth into a sterilie inoculum flask having a side arm(total volumeabout 5 liters).

Fermentation stage.-Aseptically transfer the 5-liters of inoculum to a35 gallon fermenter containing 90 liters of the sterile growth mediumemployed in the germination stage. Acrobically ferment for 20-30 hours(until the packed coil volume (PCV) is about 20-30%as determined bycentrifuging a 10 ml. sample at 2800 r.p.m. for 5 minutes) under thefollowing conditions:

Temperature C 35 Sterile air input cubic feet/minute-.. 5.4 Pressurep.s.i 7 Agitation r.p.m 180 When the PCV reaches a level of at least 2ml., aseptically transfer the contents of the fermenter to a 675 gallonformentation tank containing 450 gallons of sterile medium having thefollowing composition:

Yeast extract kg 8.5 Fish solubles kg 1.7 Corn steep liquor (dry) kg 1.7Calcium carbonate kg 1.7 Lactose kg 51.0 Anti-foam (GE60) ml 500 Softwater gallons 450 Ferment at 35 C. while agitating at 120 r.p.m. andintroducing air at 7 p.s.i. in. and 15 cu. ft./min. for 5070 hours.

At the end of this period, the potency of the produced antibioticreaches a peak which remains substantially constant as determined bysampling and assay against S.

aureus.

During the fermentation the pH remains substantially in the range of7.07.3. The packed cell volume reaches a constant value of about 2.0 ml.

EXAMPLE 2 Laboratory fermentation of M. Carbortacea Germination.rtage.-Perform the germination stage as described in Example 1 (thecalcium carbonate may be omitted).

Fermentation stage.Transfer a 25 ml. inoculum from the germination stageto each of four 2-liters flasks each containing 500 ml. of the followingmedium:

Yeast gm Fish solubles gm 1 Corn steep liquor (dry) gm 1 Calciumcarbonate gm 1 Lactose ..grn 30 Tap water ml 1000 Incubate the flasksand their contents for 23 days at 26 C. on a rotary shaker. Pool thecontents of the flasks. Work up as described in the following examplesmaking appropriate adjustments as to quantities and volumes.

EXAMPLE 3 Extraction of the antibiotics after tank fermentation Add 25kg. of filter-aid (Celite) to the 500 gal. of fermentation broth fromExample 1, agitate, and filter. Discard the mycelial cake. Extract thefiltrate twice with an equal volume of toluene. Combine the tolueneextracts and concentrate in vacuo to dryness (oily dark brown residue).Suspend the residue in 1000 ml. of chloroform, filter and add thechloroform solution under vigorous agitation to ten volumes of petroleumether. Continue stirring for five minutes. Separate the semi-solid oilyprecipitate from the supernatant liquid by decantation. Redissolve theprecipitate in 600 ml. chloroform and add the solution to five volumesof petroleum ether while stirring vigorously. Filter the mixture, washthe amorphous precipitate comprising antibiotic substances withpetroleum ether and air dry. Yield: 12.6 g. (Minimum inhibitoryconcentration against S. aureous: 15 g./ml.)

EXAMPLE 4 Separation of the antibiotics Separation of the antibioticsubstances obtained in Example 3 is etfected by partition chromatographyutilizing a two-phase solvent system and a purified diatomaceous earth(Chromosorb W, Johns Manville and Company, U.S.A., non-acid washed, meshsize 60-100) as the inert support for the heavier phase as follows:

Prepare a two-phase solvent system having the following composition:

Parts Petroleum ether (60-90 C.) 25 Ethyl acetate 75 Methyl alcohol 70Distilled water 30 Equilibrate 1.9 kg. of the inert support with 950 ml.of the heavier phase of the above two-phase solvent mixture and pour themixture slowly into a 9 cm. diameter glass column provided with afritted disc at its bottom, said column previously filled to threequarters of its length with the lighter phase. Allow the diatomaceousearth to settle in the column to a compact bed and then further compressunder a nitrogen pressure of 1 to 2 lbs/sq. inch. (The height of thecolumn thus prepared is 137 cm.)

Dissolve 7.0 g. of the amorphous matter from Example 3 in 38 ml. of theheavier phase of the two phase solvent system. Add 19 g. of thediatomaceous earth and add the solid mixture to the top of the columnbed and firmly compress. Elute with the lighter phase of the two-phasesolvent system. Collect eluates at a rate of 50 ml./min. The number ofindividual fractions is optional but related to the column hold-upvolume (HUV); HUV being defined as the intersticial volume occupied bythe lighter phase of the two-phase system in the total bed volume of thecolumn. In this column, the HUV is about 3500 ml. Discard the firsthold-up volume collected. Thereafter, chromatograph a sample of eachcollected fraction on paper using solvent system I, describedpreviously, and bioautograph the developed and dried papergram againstS. aureus. Combine fractions according to their paperchromatographicpattern. Concentrate the combined fractions to dryness in vacuo, weigh,and against paperchromatograph and bioautograph to determine therelative composition of the particular fraction combination.

By the column chromatographic separation described above, the followingresults are obtained:

TABLE IV Chromatography in Diatomaeeous Earth Paper Weight ofbioautographio Eluant, ml. residue, mg. pattern, RF

Chromatograph one gram of the material obtained from HUV 3-10 utilizingthe inert support (Chromosorb W) for the heavier phase of the followingtwo-phase solvent system:

Parts Petroleum ether (B.P. 30-60 C.) 10 Toleuene 200 n-Butyl alcohol 15Ethyl alcohol l Distiled water 7-0 Equilibrate 600 g. of thediatomaceous earth support with 300 ml. of the heavier phase of theabove two phase solvent mixture and slowly pour into a 5 cm. diam. glasscolumn partly filled with the lighter phase of the above two-phasesolvent system. Permit the support to settle by gravity to a compact bedand further compress by a nitrogen pressure of 1-2 lbs/sq. inch. Theheight of the column thus prepared is 107 cm. and its hold-up volume is900 ml. Dissolve the material to be chromatographed in 10 ml. of theheavier phase of the above two phase solvent system, add 5 g. of theinert support and mix. Add the solid mixture to the top of the columnbed and firmly press down. Perform elution with the lighter phase of theabove two phase solvent system at a flow rate of 100 ml./min.Continuously collect fractions of 50-900 ml. (corresponding toone-eighteenth to one times the holdup volume of the column bed).Discard the first hold-up volume obtained immediately after applying thetest material; start collection with the second hold-up volume.

Immediately chromatograph a sample of each col lected traction on paperusing solvent system I described TABLE V Chromatography of HUV 3-10Paper Weight of bioautographic Eluant, ml. residue, mg. Pattern, RE

EXAMPLE 5 of everninomicin D component Purification Suspend 100 mg. ofthe residue obtained from HUV 1.5-14 in the previous example in 40 ml.of ether by stirring vigorously at room temperature. Filter and chillthe filtrate overnight. Filter the precipitate which forms and wash withice cold ether. Air dry obtaining about 81 mg. of a nearly colorlesspowder, assaying at 1155 units/ mg., M.P. 138-140 C. Further purify bydissolving in a minimal quantity of hot isopropyl alcohol, adddecolorizing charcoal (amount equal to of everninomicin D), filter andcool the filtrate in an ice bath. Filter the precipitate, wash with coldisopropyl alcohol and dry in high vacuum at room temperature.Everninomicin D so prepared assays at 1450 units/mg.

The purified everninomicin D obtained above is biologically homogeneouswhen assayed qualitatively against S. aureus. When the everninomicin Dis chromatographed on a thin layer of silica gel (Stahl technique) usingacetone-benzene (1:1) as the eluting agent followed by sulfuric acidtreatment of the developed, dried plate, so other substance but the Dcomponent is detectable.

EXAMPLE 6 Acetylation of everninomicin D Dissolve 25 mg. everninomicin Din 1 ml. of pyridine. Add 0.4 ml. acetic anhydride and allow the mixtureto stand overnight. Filter the precipitate, wash with water to removepyridine and acetic acid, dry over phosphorous pentoxide in high vacuumovernight. Yield: 11 mg. colorless amorphous powder, M.P. 135136 C.Isolate a second crop of material from the mother liquor of the aboveprecipitate by cooling the solution overnight in a refn'geratr (5 C.),filtering the precipitate, washing it free from any pyridine and aceticanhydride and drying it as above. Yield: 6 mg. of a colorless amorphouspowder. Purify by dissolving the crude acetylation product in methylenechloride and adding thereto decolorizing charcoal. Filter and add hexaneto the clear filtrate. Filter the precipitate and airdry.

When chromatographed on thin layer, using a silica gel, (Silica Gel G.E.Merck A.G., Darmstadt, Germany) as adsorbent and acetone: benzene (1:1)as eluting system and upon spraying of the developed and dried platewith sulfuric acid there is obtained a single component of R value 0.87.(Under these conditions the D component has an R, value of 0.49). Uponalkaline hydrolysis in methanol, everninomicin D is regenerated.

EXAMPLE 7 Methyl ether of everninomicin D Dissolve 500 mg. everninomicinD in 11 ml. of ethyl acetate and 11 ml. of ethyl ether. Add an, excessof an ethereal solution of diazomethane and let the yellow solutionstand at 5 C. for 72 hours. Evaporate the solution to dryness under astream of nitrogen. Dissolve the crude product in 5 ml. ofisopropylalcohol, refrigerate and collect the precipitate by filtration.Wash with cold isopropyl alcohol and dry obtaining 260 mg. of the methylether as colorless prisms, M.P. l61l62 C.; [a] =-17.7 (1% in pyridine);A 288 m (E =13.7).

EXAMPLE 8 Purification of everninomicin E This non-polar component ofthe antibiotic mixture is usually found in high concentration in thesupernatant liquor of the chloroform-petroleum ether precipitation ofthe crude fermentation extract described in Example 3. It also ispresent, to a lesser extent, in the precipitated antibiotic substancesand is recoverable therefrom by utilizing the column partitionchromatography method described in Example 4.

In order to obtain everninomicin E, concentrate HUV 11.5, from Example4, to dryness in vacuo and air dry. There is obtained about 320 mg. ofan amber-colored material which is further purified by precipitationfrom etherhexane yielding 280 mg. of a nearly colorless amorphous powderrepresenting purified E component.

Upon paper chromatography in System I and .bioautography against S.aureus and B. subtilis, Everninomicin E is characterized as beingbiologically homogeneous.

EXAMPLE 9 Isolation of everninomicin B Isolation of the B component iseffected by similar partition chromatography as described in Example 4by utilizing a two-phase solvent system and a purified diatomaceousearth (chromosorb W. Johns Manville and Company, USA, non-acid washed,mesh size 60-100) as the inert support for the heavier phase as follows:

Prepare the two-phase system (petroleum ether, ethylacetate, methanol,water) described in Example 4.

Equilibrate 2.5 kg. of the invert support with 1250 ml. of the heavierphase of the two-phase solvent mixture and prepare the column bed asdescribed in Example 4.

Dissolve 7.4 g. of the amorphous precipitate from Example 3 in 40 ml. ofthe heavier phase of the two-phase solvent system. Add 20 g. of thediatomaceous earth and add the solid mixture to the top of the columnbed and firmly compress. Elute with the lighter phase of the twophasesolvent system. The HUV of this column is about 8000 ml.

By following the column chromatogrphic separation procedure of Example 4the following results are obtained:

TABLE VI.RESOLUTION OF ANITBIOIIC SUBSTANCES BY CHROMATOGRAPHY ONDIATOMACEOUS EARTH Weight of Paper bioautographie Eluant, ml. residue,mg. pattern, Rr

1 8,000 0, 0.16; 0.23; 0 55 (trace) 1 Acetone elution. 2 50% acetone inmethanol elation.

Chromatograph 1.8 gram of the material obtained from HUV 1.9-2.8 on 1800grams cellulose powder (Whatman, non-acid washed; fine grade) packedinto a column of 7.5 cm. diameter to a height of cm. by using thefollowing one phase solvent mixture.

Parts Acetone 1 Petroleum ether (B.P. 30-60 C.) 25 Benzene 50 Dissolvethe material to be chromatographed in 68 ml.

of the solvent mixture, admix 50 g. cellulose and charge the mixtureto'the top of the column bed. Perform elution at a flow rate of 50ml./rnin. Continuously collect fractions of 100 ml. The HUV of thiscolumn is about 6000 ml. Combine eluates as shown in the followingtable.

solution to 70 ml. of ether-hexane mixture to 4 v./v.) with vigorousstirring. Filter the precipitate formed. Evaporate the filtrate in vacuoto small volume (approximately 20 ml.) and separate the percipi'tateformed by filtration. Evaporate the filtrate in vacuo to dryness toobtain purified component F as an oil-white powder.

CHEMICAL AND PHYSICAL PROPERTIES Everninornicin B Alternatively,everninomicin B is separated from ever- 10 Eveminomicin B, Produced bythe method of ninomicin D and E by chromatography on Fl i jl Examples 9and 10 is a colorless substance having the tivated -magnesium silicate,Floridin Company Tallafollowlng Propeftles: hassee, 1 as l (I) Meltingpoint: (Koefiler block): l54-157 C.

Prepare a bed of 22.5" height and 1.75" diam. by pour- Optical ati n [a]=25.5 (c.=1% in P ing a methylene chloride slurry of 500 grams ofFlorisil 60-400 mesh, activated for 18 hours at 105 C., into aUltraviolet absorption: max 233 in glass column. The hold-up volume ofthe bed is 1100 ml. methanol) max 296 i in 011 N methanolic Dissolve 50g. of crude antibiotic amorphous precipitate (obtained in Example 3) in300 ml. of methylene chlo- 20 Analysis:

ride and add the clear solution to the Florisil column. Elemental! Eluteby passing four hold-up volumes of methylene chlo- C=51.02%

ride, followed by eight hold-up volumes of methylene chlo- H=6.68%

ride containing 5% acetone, eight hold-up volumes of N=1.23%

methylene chloride containing 10% acetone, 8 hold-up 0:34.72%

volumes of methylene chloride containing 20% acetone, Cl=3.9'7%

8 hold-up volumes of methylene chloride containing 50% (b) Functionalgroups:

acetone, and finally 16 hold-up volumes of pure acetone. OCH =12.80%

The results of such a procedure are set forth in the (C)CH =7.15%following table: (N)CH =2.33%

TABLE VIII.-FLORISIL CHROMATOGRAPHY OF CRUDE ANTIBIOTICS Paper biauto-Weight of graphic Eluant Eluate, ml. residue, g. pattern; R;

011 01 4, 400 10. 0.43m); 0.56(E) OHZCIZ plus 5% acetone- 8, 800 8. I0:45(D) CHQCP plus 10% acetone- 8, S00 13. 2 0.43(D) 011 01 plus 20%acetone. 8,800 2.3 0.45(D); 024(3) CH2O12 plus 50% acetone 8,800 6. 30.2 B) 100% acetone 8, 300 5. 4 0.150); 0.2303) In the foregoing table,the latter designate in the right-hand column denotes the particulareverninomicin component present in the particular eluate.

EXAMPLE 10 Purification of everninomicin B Dissolve 200 mg. of theresidue obtained from HUV 17.7-27.7 obtained from cellulose column ofExample 9 in 5 ml. of acetone. With vigorous agitation add the acetonesolution to 50 ml. of hexane. Filter the precipitate which forms andwash with hexane. Dry at room temperature in vacuo over phosphoruspentoxide obtaining about 120 mg. of an off-white powder.

Further purification of component B from either technique of isolationis achieved by dissolving the amorphous powder in hot isopropyl alcohol,adding 10% by weight decolorizing charcoal, stirring for 10 minutes,filtering and cooling the filtrate to 5 C. The precipitate is collectedby filtration, washed with cold isopropyl alcohol, and dried in highvacuum at room temperature for 24 hours. It assays at 760 units/ mg.

The filtrate s from the above purification are combined and employed inExample 11 below for the isolation and purificaton of everninomicin F.

EXAMPLE l1 Isolation and purification of everninomicin F Evaporate todryness in vacuo the combined filtrates from Example 10. Dissolve theresidue (422 mg.) in acetone (7 ml.) and filter oli insolubles. Add theacetone (V) Infrared spectrum: (See FIGURE 1 for spectrum in mineraloil--Nujol.) The absorption peaks are located at the followingwavelengths (with peak strength codified as follows: W=weak, M=moderate,M-S: moderate to strong, s strong, VS=very strong, sh:

shoulder):

A (,u)! Park strength 6.10 M 6.35-6.39 m (doublet) 6.45 S

7.40 sh 7.67-7.80 sh 8.12 sh 8.308.3,5 S (broad) 8.55 sh 8.75-9.80 S(broad) 10.20-40.26 S (broad) 10.30 sh 13 kmax, (,u.) Peak strength10.85 sh 11.03 M 11.52. M

11.70 sh 11.85-12.00 sh 12.75-12.81 W (broad) 12.98 W 13.54-13.60 M(broad) 13.85-13.90 M (broad) insoluble in ether, hexane and water.

Everninomicin D The antibiotic, as produced herein, is a white amorphouspowder having the following physical and chemical characteristics: (I)Melting point: (Koefiler block): 138-l40 C., from ether trituration;after recrystallization from isopropanol, M.P.=160-161 C. (II) Analysis:

(a) Quantitative:

C=51.79% H=6.35% N=1.40%

O=36.35% C1=3.98% OCH =13.30% C(CH )=6.93% N(CH )=1.98% (b) Qualitative:

(1) Ninhydrin test-negative (2) Elson-Morgan testpositive (3) AlkalineKM O testpositive (4) Anthrone test--grayish blue (5)2,4-dinitrophenylhydrazine test: positive (6) Ehrlich (diphenylaminetest): negative (7) Triphenyltetrazolium test: weakly positive (III)Rotation: [oz] =25.3 (1% in methanol),

-37.7 (1% in pyridine). (IV) Ultraviolet spectrum: )r at 289 m (e =22).

(In a solution of 6 ml. of 1.0 N potassium hydroxide in 100 ml. ofmethanol, )t shifts to 295 m (e =79, methanol).) (V) Infra-red spectrum:(See FIGURE 2 for spectrum in mineral oil-Nujol). The absorption peaks(W=weak,

=moderate, M4=moderate to strong, S:strong, VS=very strong, sh=shoulder)are located at the following wavelengths:

A (,u.) Peak strength 2.89 S 5.73 S 6.12 W 6.35 sh 6.45 S 7.10 I sh7.30' sh 7.42 S

7.68-7.84 S (broad) 7.99 -1; S 8.33 S

8.48-8.70 sh 8.84-9.17 S (broad) 9.57 -1. S (broad) 10.00 L sh10.22-10.26 S (doublet) 10.55 S

10.91 sh 11.00 M

14 maxil) i Peak strength 11.50-11.60 M (broad) 11.66 Sh 11.90-12.00 M(broad) 12.25-12.35 W (broad) 12.70-12.85 W (broad) 13.00 W

13.52-13.60 W (broad) 13.85-13.90 W (broad) 14.42-14.58 W (broad) (VI)Solubility:

Very soluble in: chloroform, methylene chloride, acetone and methanoland 0.1 N sodium hydroxide.

Sparingly soluble in ether.

Insoluble in: petroleum ether, toluene, benzene, water 10% sodiumbicarbonate (aqueous) and 10% sodium carbonate (aqueous).

(VII) Stability:

The antibiotic is stable at 0 C. and in the dark.

In methanol solvent containing a trace of pyridine, it

is stable for at least two weeks.

Rapid deactivation occurs at a pH below 5.5 whereas at pH above 7 and upto 12 the activity is retained for a few days.

Upon treatment with 0.1 N methanolic HCl at room temperature for 16hours the antibiotic activity is destroyed. Treatment of the acidicmixture with a stoichiometric amount of sodium bicarbonate (2% aqueoussolution), followed by removal of the methanol by concentration in vacuoand extraction with chloroform yields a mixture of five component asdetermined by thin layer chromatography on silica gel using benzeneacetone (75:25) as a solvent and sulfuric acid as a reagent spray. Allcomponents are less polar than everininomicin D.

Upon treatment with 1.0 N NaOH at room temperature for 16 hours theactivity of component D is essentially unchanged.

(VIII) Derivatives:

(a) Acetate-prepared as in Example 6 M.P. 151-160 C., U.V. absorptionmaximum at 286 m (e k-9:2). R0tation-[u] =25.4 (1% in methanol).Analysis: C=53.08%, H=7.08%, N=0.77%. Infrared (Nujol) absorption bandsat 2.85, 5.57, 5.71, 6.14, 6.45, 8.02, 8.38, 8.88, 9.17, 9.55

Everninomicin E Component E, as isolated and purified, as describedheretofore, is a nearly colorless, amorphous powder having the followingphysical and chemical properties:

(I) Melting point: 89-96 C. (II) Analysis:

(a) Quantitative O=65.l7% H=7.54% N: 1.3 1% 0:24.25

Qualitative (1) Ninhydrin testnegative (2) Elson-Morgan-positive (3)Alkaline KM O --positive (4) Anthronepositive (5)2,4-dinitrophenylhydrazine-positive .(6) Ehrlich (diphenylamine)negative(7) Triphenyltetrazoliumpositive (III) Rotation M1 +36.7 (1% indioxane).(IV) Ultraviolet spectrum: )r 240m (methanol),

(e =232). '(V) Infrared Spectrum: (See FIGURE 3 for spectrum in mineraloil-(Nujol). The absorption peaks are located at the followingwave-lengths with intensity indicated as before:

(VI) Solubility:

Component E is soluble in: chloroform, methylene, chloride, acetone,methanol and ether. Partially soluble in benzene, toluene and hexane.Insoluble in water.

Everninomicin F Component F, isolated and purified as describedheretofore, is an off-white amorphous powder having the followingphysical and chemical properties:

(I) Melting point: 9l98 C.

(II) Analysis:

(a) Quantitative Qualitative l) Ninhydrin testnegative (2)Triphenyltetrazolium-positive (III) Rotation: [a] =24.3 (1% in dioxane).

(IV) Ultraviolet Spectrum: A 240 m (methanol) (V) Infrared Spectrum: Theinfrared spectrum was run on an evaporated film and showed absorptionpeaks located at the following Wave lengths with intensity indicated asbefore. (See FIGURE 4 for spectrum from which the following absorptionpeaks are derived):

k (lL): Peak strength 2.85 M 3.373.41 S 5.77 S

5.83 sh 5 90 M-S 6.17 M-S 6.83 M

7.22 M 7.58 M 7.80 M 7.97 sh 8.42 M-S 8.57 M-S 899 M-S 9 M-S 9.53 M-S9.75 M

M Peak strength (VI) Solubility:

Everinomicin F is soluble in most organic solvents including:chloroform, methylene chloride, acetone methanol, ethanol, ether,benzene, toluene and hexane.

Insoluble in water.

BIOLOGICAL PROPERTIES OF ANTIBIOTICS PRO- DUCED BY THE FERMENTATION OFM. CARBONACEA The antibiotics produced as described herein, particularlyeverninomicin components B, D and F exhibit a broad range ofanti-microbial activity against gram-positive pathogenic microorganisms.Components B, D and F are of particular value in combatting infectionsproduced by penicillin-resistant microorganisms such as certain strainsof Staphylococcus aurcus and are of value in combatting certainmicroorganisms which are susceptible to destruction by the penicillins.It is known that many disease manifestations are caused by gram-positiveorganisms (such as Streptococcus, Staphylococcus, Pneurnococcus, and thelike). These are properly controlled and treated by means of the actionof the antibiotics described herein, principally everninornicin B,everninomicin D and everninomicin F. A particular manifestation isinfectious bovine-mastitis which is generally caused by species ofStaphylococcus (aureus) and Streptococcus (agalcnctiae, dysgalacliae,and uben's). These antibiotics effect esentially complete cure of thedisease after a relatively brief regimen of administration. Further highpotency and effect has been elicited against pathogenic avian strains ofpleuropenumonia-like organisms and accordingly these antibiotics are ofvalue to chicken breeders and egg farmers.

In addition to it activity against gram-positive microorganisms,everninomicin F, unlike the other antibiotics described herein, is alsoeffective against a broad range of pathogenic microorganisms includingspecies of the genus Escherichia, Salmonella, Proteus and Psuedomonas.It is well known that many serious disease syndromes including urinarytract infections and diarrhcas are caused by gram-negative organisms.These syndromes, which are quite common in domestic animals such ascattle, horses, sheep, swine and household pets such as dogs and catsmay be effectively treated and controlled by means of the action ofeverninomicin P which usually olfers a prompt and complete cure.

The gram-positive only antibiotics produced herein by virtue of theirantibacterial action against microorganisms such as Staphylococcusaureaus, Bacillus subtilis and the like are advantageously employed aslaboratory reagents when attempting to determine the presence ofgram-negative organisms. They may be used to inhibit overgrowth of suchorganisms in culture media, either alone or in combination with otherantibacterial agents to reduce or eliminate the heavy overgrowth ofgram-positive organisms permitting the determination of gram-negativeorganisms such as Klebsiella penumom'ae or Escherichia coli in culturesobtained in diagonstic procedures. As such reagents they may be employedin solution such as in alcohol. Everninomicin F, by virtue of itsactivity against gram-positive and gram-negative microorganisms, may beused to supress bacterial growth in culture media permitting thedetermination of yeasts and molds in diagnostic cultures. In view oftheir action against grampositive organisms, the antibiotics describedherein may be used to sterilize equipment such as in operating rooms inhospital Wards,

17 The comparative in vitro activities of everninomicin B (as producedin Example 10) everninomicin D (as produced in Example and everninomicinF (as produced in Example 11) are set forth in Table IX below.

The invitro activity of everninomicin F against gramnegativemicroorganisms is shown in Table IX(A) below. The susceptibility of thetest microorganisms to the antibiotics was determined by standard tubedilution methods. In each instance, dilutions of 24 hour broth cultureswere employed as inoculum with the end points being taken afterincubation for 24 hours at 37 C. in a Difco Penassay broth medium (DifcoLabs, Detroit, Mich). In the tables, the activity of the respectiveantibiotics is expressed in units per milliliter or micrograms permilliliter, a unit being as defined heretofore.

TOXICITY IN MICE OF EVERNINO- TABLE XI.ACUTE MICINS B AND D LD/50(mg/kg.)

Mode of Administration B D Subcutaneous 2, 500 1, 000 r Intraperitoneal750 500 TABLE IX.-IN VITRO ANTIBIOTIC SPECTRUM OF EVERNINOMICINCOMPONENTS B, D AND F Minimal Inhibiting Concentration TestMicroorganism (Units/ml.) (Units/ml.) g/ml.)

Streptococcus pyogeues 2 DA 21- 0.15 Streptococcus hemolyticus 0. 1Streptoc ccus faecalts ATC C 10541 0. 3

1 DA refers to strain identity in private collection of ScheringCorporation, Bloomfield,

New Jersey.

Brain-heart infusion broth plus 0.5% of human serum.

Table IX(A).-In vitro antibiotic spectrum, gramnegative) everninomicin FMinimal inhibiting Test microorganism: concentration ,ug./ ml.)

Escherichia coli ATCC 10536 Klebsiella pneumoniae DA 20 7.5 Proteusvulgaris DA 121 15.0 Pseudomonas acruginosa ATCC 8689 15.0

The in vivo activity of everninomicin B and D has been elicitedpharmacologycally in a standard test animal (mouse 18-20 g.) againstcertain pathogenic microorganisms. The standard test procedure employedis as follows. Thirty mice were infected with an inoculum of theparticular pathogen adminstered by intraperitoneal injection. Twentymice were then treated by subcutaneous injection of the antibioticdissolved in ethanol (2 parts), Tween 80 (0.5 part), peanut oil (8.5parts) and the injection administered in two equally divided dailydosage. Ten mice were maintained as controls, that is, untreated. Allcontrol animals died within 18 hours. The protecting dose relative tosurvival of of the treated animals (PD/ 50) for 48 hours was evaluatedto be as follows in conjunction with the particular pathogen.

TABLE X.IN VIVO ACTIVITY OF EVE RNINOMICIN COM- PONENTS B AND DSalmonella schottmuelleri DA 10 PD/50 (mg/kg.)

The in vivo activity of everninomicin F has also been confirmed in miceemploying standard pharmacological The in vitro activity ofeverninomicin E against representative microorganisms is set forth inTable XII below.

TABLE XII.-IN VITRO ACTIVITY OF EVERNINOMICIN E Size of Zone ofInhibition mm. against Concentration Component E ugJml. S. aureus S.fecalis B. subslilz's It will be noted that the antibiotics as describedabove, with the exception of component E, are chlorine containingantibitoics. Fermentation of Micromonospora carbonacea in the chlorinecontaining media described here tofore permits the biosyntheticutilization of chlorine introduced into the fermentation media from suchsources as the yeast extract, fish solubles and water employed in itspreparation. From the foregoing, however, it will be obvious to oneskilled in the art that modification of the halogen content of thefermentation medium can. be effected with consequent modification of thehalogen content of the antibiotics produced.

It will be obvious, for example, that deschloro derivativescorresponding to the chlorinated antibiotics described above can beprepared by fermenting Micromonospora carbonacea in fermentation mediumas heretofore described wherein the chlorine content has beensubstantially reduced or eliminated. Chlorine can be removed from themedium by silver nitrate precipitation and/ or by removal on anionicexchange resins. Reduction of chlorine content in the fermentation mediacan also be effected by the addition thereto of organic chlorinationinhibitors such as mercaptobenzothiazole. Applicants consider thedeschloro derivatives so produced to be the full equivalents 19 of theantibiotics described above and to fall within the scope of thisinvention.

Further, it will be obvious to anyone skilled in the art that bromoderivatives corresponding to the chlorinated antibiotics described abovecan be prepared by fermenting Micromonospora carbonacea in a mediumcontaining bromine. Such bromine containing medium is readily obtainedby adding bromine containing compounds such as, for example, sodiumbromide or potassium bromide to a medium dechlorinated as previouslydescribed, Applicants consider the bromo derivatives prepared by suchfermentation also to be the full equivalents of the chlorinatedantibiotics described herein and to fall within the scope of thisinvention.

We claim:

1. A method for producing everninomicin which comprises cultivating amicroorganism selected from the group consisting of Micromonosporacarbonacea var. carbonacea NRRL 2972 and Micromonospora carbonacea var.aurrmtiaca NRRL 2997 under submerged aerobic conditions in an aqueousnutrient medium containing an assimilable source of carbon and nitrogenuntil a composition of matter having substantially antibiotic activityis produced and recovering said antibiotic composition therefrom.

2. A method according to claim 1 wherein the organism in Micromonosporacarbonacea var. carbonacea NRRL 2972.

3. A method according to claim 1 wherein the organism is Micromonosporacarbo'nacea var. aumntiaca NRRL 2997.

4. A method for producing antibiotics everninomicin B, everninomicin D,everninomicin E and everninomicin F by the process of claim 1 in anaqueous chlorine containing nutrient medium under submerged aerobicconditions until a composition of matter having substantial antibioticactivity is produced and recovering said antibiotics therefrom.

5. A method according to claim 4 wherein after substantial antibioticactivity is produced, the mycelium is separated from the medium and thebroth is extracted with an organic solvent from which the antibioticcompositons of matter are isolated.

6. A composition of matter, identified as antibiotic everninomicin B,effective in inhibiting the growth of gram-positive bacteria, saideverninomicin B being an organic substance having the followingelementary analysis: C=51.02%, H=6.68%, N=1.23%,

and Cl:3.97%, that has a specific optical rotation as measured by the Dline of sodium at 25 C. of 25.5 in pyridine at 1% concentration, isinsoluble in water, ether, and hexane, is slightly soluble in benzeneand very soluble in halogenated hydrocarbons, acetone, methanol,ethanol, pyridine and dilute aqueous alkali; has an ultravioletabsorption maximum at 288 m with 6 in methanol equal to about 12; has aninfrared absorption when suspended in solid form in hydrocarbon oilsubstantially as shown in FIGURE 1; has a melting point of about154-157" C.; and has an antibacterial spectrum including the bacteriaenumerated in Table IX.

7. A composition of matter, identified as antibiotic everninomicin D,effective in inhibiting the growth of gram-positive bacteria, saideverninomicin D being an organic substance having the followingelementary analysis: C=51.79%, H=6.35%, N=1.40%

and Cl=3.98% that has a specific optical rotation as measured by theD-line of sodium at 25 C. of -37.7 in pyridine at 1% concentration and25.3 in methanol at 1% concentration; is soluble in halogenatedhydrocarbons, acetone, methanol, and dilute aqueous alkali, is sparinglysoluble in ether and insoluble in water, petroleum ether, toluene andbenzene; has an ultraviolet absorption maximum at 295 mg with e inmethanol equal to about 79; has an infrared absorption spectrum whensuspended in solid form in hydrocarbon oil substantially as shown inFIGURE 2; has a melting point of about 16l C.; and has an antibacterialspectrum including the bacteria set forth in Table IX.

8. A composition of matter, identified as antibiotic everninomicin E,effective in inhibiting the growth of gram positive bacteria; saideverninomicin E being an organic substance and characterized in havingan infrared spectrum when suspended in solid form in hydrocarbon oilsubstantially as shown in FIGURE 3 and further characterized by thefollowing parameters: Analysis: C=65.l7, H:7.54, N=l.3l, 0:24.23;soluble in chloroform, methylene chloride, acetone, methanol and ether,sparingly soluble in hexane, benzene and toluene; insoluble in Water,ultraviolet absorption maximum at 240 m with e in methanol equal toabout 232; [m] =+36.7 (1% in dioxane); melting point 89-96 C.;qualitatively negative in the ninhydrin test, Ehrlich (diphenylaminetest); qualitatively positive in the Elson-Morgan test; alkalinepermanganate test; anthrone test, 2,4-dinitrophenylhydrazine test, andtetraphenyltetrazolium test.

9. A composition of matter, identied as antibiotic everninomicin F,eifective in inhibiting the growth of gram-positive and gram-negativebacteria, said everninomicin F being an organic substance having thefollowing elementary analysis; C=60.68%, H:8.85%, N=2.41%, O=26.96% andCl:l.l0%; that has a specific optical rotation as measured by the D-lineof sodium at 25 C. of -24.3 in dioxane at 1% concentration; is solublein organic solvents including chloroform, methylene chloride, acetone,ethanol, methanol, ether benzene, toluene and hexane and is insoluble inwater; has an ultraviolet absorption maximum at 240 m with 6 in methanolequal to about 206; has an infrared absorption spectrum as an evaporatedfilm substantially as shown in FIGURE 4; has a melting point of about91-98 (3.; and has an antibacterial spectrum including the bacteriaenumerated in Table IX and Table IX(A).

References Cited Derwewt Farmdoc 15,634, Neth. Patent 6,408,506,published Jan. 25, 1965, pp. 339-375.

ALBERT T. MEYERS, Primary Examiner JEROME D. GOLDBERG, AssistantExaminer US. Cl. X.R.

mg UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. uggma Dated March 5, 131;)

Inventor(s) George M. Luedemann and Marvin J. Weinstein It is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Column 1, line 19, "bang" should read --being--. Column 1, line 42, "iswash" should read --in wash--. Column 1, line 55, "variable" should read--viable--. Column 2, line 8, "visible culture" should read --viahleculture--. Column 2, line +3, "Coloney" should read --Colony--. Table I"Type A, 1%" should read "Type A, l$--. Column 6, line 66, "packed coil"should. read --packed cell. Columnl6 and 7, line 75 and l,"fermentation" should read --fermentation--. Column 8, line 28, "andagainst" should read --and again--. Column 8, line 51, toleuene" shouldread --Boluene--. Column 8, line 5k "Distiled water" should readDistilled water--. Column 9, line 1-0, "dried plate, lo" should read--dried plate, no--. Column 9, line 55, "refrigeratr" should read--refrigerator--. Column 9, line 66 "and B value" should read --an Rvalue". Column 10,

line 52, "pattern, R should read --pattern, R Column 10,

line 57, "0 55 (trace)" should read --0.55 (trace)--. Column 11, line"pattern, Rf should read --pattern, R Column 11,

line 3 "pattern; R should read --pattern; R Column ll, line 1-5 "latterdesignate" should read --letter designate--. Column 12, line 52 "Parkstrength" should read -Peak strength--. Column 1 1, line 33 "fivecomponent" should read --five components--. Column 1 1, line 38"evenninomicin" should read --everninomicin--. Column 1L, line 63"O-2h.25$" should read --0-2 +.23$--. Column 15, line 3 1 max 1) shouldread R (;1)--. Column 16, line 36 "esentially" should read--essentially--. Column 17, Table III, line ll, "Staphylococcus auruseSmith" should read --Staphylococcus aureus Smith--. Column 17, line 52"pharmacologycally" should read --pharma cologically-. Column 17, 111260"daily dosage" should read --daily dosages--. Column 19, claim 1 "havingsubstantially" should read ---having substantial--. Column 19, claim 6"o-53h.72$" should read --o-5u.72$--.

3m?? Aflu sFzlFa SEP 151970 (SEAL) Attest:

Edward m. Fletcher, Jr. mm

Omissioner of Patents Attesfing Officcr

